Severe progressive form of congenital muscular dystrophy with calfpseudohypertrophy, macroglossia and respiratory insufficiency

Susana Quijano-Roya, Lucıa Galanb, Ana Ferreirob, Fawzia Cheliout-Herautc, Francoise Grayd,Michel Fardeaub, Annie Baroisa, Pascale Guicheneyb,*, Norma B. Romerob, Brigitte Estourneta

aService de Pediatrie, Reeducation et Reanimation Neurorespiratoire, Hopital Raymond-Poincare, 92380 Garches, FrancebINSERM U523, Institut de Myologie, IFR ‘Coeur, Muscle et Vaisseaux’ N.14, Groupe Hospitalier Pitie-Salpetriere,

Batiment Babinski, 47 Bd. de l’Hopital, 75013 Paris, FrancecLaboratoire d’Explorations Fonctionnelles, Hopital Raymond-Poincare, 92380 Garches, France

dLaboratoire de Neuropathologie, Faculte de Medecine Paris-Ouest, 92380 Garches, France

Received 21 June 2001; received in revised form 18 November 2001; accepted 22 November 2001

Abstract

A novel form of congenital muscular dystrophy in four unrelated patients is proposed. Congenital hypotonia, markedly increased CK, calf

pseudohypertrophy and proximal weakness were common early findings. Two cases were severely affected since infancy and never walked.

The phenotypical homogeneity was not very evident until advanced stages of the disease. All the patients showed catastrophic progression of

the weakness, severe restrictive respiratory insufficiency, macroglossia, peculiar extreme amyotrophy of hands and feet, and a round and

‘puffy’ face. All patients became tetraplegic and required mechanical ventilation. Two cases had signs of mild cardiac involvement. The only

non-tracheotomised patient died of respiratory complications. No mental retardation or specific brain abnormalities were observed. All

patients showed secondary deficit of laminin a2 and up-regulation of laminin a5 in muscle. Expression of a-dystroglycan was severely

reduced in two available muscle samples. The known loci for congenital muscular dystrophies were excluded in the only consanguineous

case by linkage analysis. Clinical, immunohistochemical and genetic findings strongly suggest a distinct entity. q 2002 Elsevier Science

B.V. All rights reserved.

Keywords: Congenital muscular dystrophy; Macroglossia; Calf hypertrophy; Respiratory insufficiency; Secondary merosin deficiency; a-dystroglycan

1. Introduction

The diagnosis of congenital muscular dystrophy (CMD)

is based on the finding of dystrophic changes in the muscle

biopsy of patients with congenital hypotonia and/or weak-

ness which appears at birth or early in life. It was not until

1991 that CMD was included in the International Classifica-

tion of Diseases (ICD). Since then, the nosological entity of

CMD has been extensively discussed and extraordinary

advances have been achieved in its characterisation, classi-

fication and diagnosis [1]. Different forms have been iden-

tified and therefore, CMDs are currently considered a very

heterogeneous group of diseases on clinical, immunocyto-

chemical and genetic grounds. Some CMDs show clinical

features exclusively or predominantly derived from the

muscle dystrophy (classical or ‘pure’ CMDs), while others

associate severe mental retardation and brain or cerebellar

malformations (Fukuyama type – FCMD, Muscle–Eye–

Brain disease – MEB, Walker–Warburg syndrome). Genetic

mapping has been achieved recently in FCMD and in MEB

disease [2,3]. These CMDs with structural central nervous

system abnormalities are very rare in occidental countries.

FCMD is found in Japanese population, while MEB disease,

the Finish form, is found predominantly in Northern

European countries. Regarding classical CMDs, about half

of the patients in occidental countries show a primary defi-

ciency of the laminin a2 chain (merosin) due to mutations in

the LAMA2 gene, located in chromosome 6q22 [4,5]. The

remaining CMD patients, with or without brain abnormal-

ities, may be divided basically in two non-specific

subgroups: those with a normal merosin staining in the

muscle biopsy (‘merosin-positive’ CMDs), and those with

a secondary merosin deficiency, not due to defects in the

LAMA2 gene (CMDs with secondary merosin deficiency).

The identification of new, well-characterised CMD forms

and their genetic analysis is essential for making advances

Neuromuscular Disorders 12 (2002) 466–475

0960-8966/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved.

PII: S0960-8966(01)00331-5

www.elsevier.com/locate/nmd

* Corresponding author. Tel.: 133-1-42-16-57-05; fax: 133-1-42-16-57-

00.

E-mail address: p.guicheney@myologie.chups.jussieu.fr

(P. Guicheney).

in the knowledge of these disorders. A number of new

CMDs with distinct phenotypes have been recently identi-

fied. CMD associated with a rigid spine syndrome and

restrictive respiratory insufficiency has been mapped to

chromosome 1p35 [6]. A form with calf hypertrophy that

shows secondary merosin deficiency in the muscle biopsy

(MDC1B) has been localised in chromosome 1q42 [7,8].

Distal hyperextensibility is seen in a proportion of patients

with normal staining to merosin and collagen VI deficiency

(Vanegas et al., 2001). Rarity and genetic heterogeneity of

CMDs makes it difficult to further progress in individual

series, except for large consanguineous families. Therefore,

description of new phenotypes, and discussion and colla-

boration in multicentric studies may be very helpful to iden-

tify more infrequent or ill-defined entities.

We report here four unrelated patients with distinct clin-

ical findings. Although early features were not specific, all

the four cases showed the same progressive pattern and a

homogeneous clinical appearance at advanced stages of the

disease, thus raising the possibility of a common origin. A

comprehensive and updated review on CMDs, other than

those derived from LAMA2 defects, is included in the report

of the 85th ENMC International Workshop on CMD [9].

Our patients were briefly mentioned in this report. More

extensive clinical, immunocytochemical, and genetic

features are presented and discussed here.

2. Patients and methods

2.1. Patients and complementary examinations

Four unrelated patients, three females (cases 1, 2 and 4)

and one male (case 3) were admitted for evaluation at

Raymond Poincare Hospital between 1990 and 1999.

Consanguinity was reported only in case 3. The reason for

the initial consultation was congenital hypotonia/delayed

motor development (cases 2, 3 and 4) or proximal weakness

(case 1). Myopathy was suspected in all the patients based

on increased CK levels (at least ten times of controls) and

myopathic changes on needle EMG with normal motor and

sensory nerve responses. Open muscle biopsies revealed

dystrophic changes in all cases. Cardiac and respiratory

functions were regularly assessed by using echocardiogram,

ECG, holter recording and pulmonary function studies in all

patients. To investigate central nervous system involve-

ment, brain MRI and auditory evoked potentials were

performed once at least in all cases. Visual and upper

limb somatosensory evoked potentials were available only

in cases 1, 2 and 3.

2.2. Light microscopic studies

Open skeletal muscle-biopsies were performed in patient

1 at seven years from the left deltoid muscle and at nineteen

years from a gastrocnemius muscle. A third sample was

obtained from paraspinal muscles during a surgical proce-

dure when the patient was 25 years old. Patient 2 had three

muscle biopsies, at the ages of 14 months, 7 years and 19

years. Patient three was biopsied twice, at ten months from

the left deltoid muscle, and at fifteen years from the tibialis

anterior muscle. Case 4 was biopsied twice from a deltoid

muscle at 6 months and 8 years of age. All samples were

snap frozen in isopentane, cooled in liquid nitrogen and

stored at 2808C. Transverse cryostat sections (10 mm

thick) were performed and stained using routine histochem-

ical methods [10]. A sample from patient 1 was also

embedded in paraffin and stained using the Congo Red

method.

2.3. Inmunofluorescence studies

Serial cryostat sections (8 mm thick) were incubated for

1 h at room temperature with monoclonal antibodies against

dystrophin (COOH terminal, Novocastra 1:10), a-sarcogly-

can (Novocastra 1:100), a-dystroglycan (Upstate 1:50), b-

dystroglycan (Novocastra 1:100), laminin a2 (MAB1922

Chemicon 1:1000; NCL-merosin Novocastra 1:100), lami-

nin a5 (Chemicon 1:500), laminin b1 (Chemicon 1:500)

and laminin g1 (Chemicon 1:500) chains. Antibodies were

visualised by incubation for 1 h in fluorescein-conjugated

antimouse goat IgG diluted 1/100 (Boehringer-Manheim).

Primary antibodies were visualised using a biotin-streptavi-

din-Texas Red method. Sections were examined with a

Zeiss Axioplan microscope fitted with epifluorescence.

Negative controls consisted of preincubation with PBS

and omission of the primary antibody. Immunocytochem-

ical studies of a-dystroglycan, b-dystroglycan, laminin a2

with the NCL-merosin antibody that recognises the

300 KDa fragment, laminin b1 and laminin g1, were not

performed in patient 1 and patient 4 due to technical

problems in the original sample conservation. Two addi-

tional samples were taken from patient 1 at 25 years for

immunocytochemistry, but no muscular fibres were found.

No new muscle samples were available from patient 4 who

died in 1997.

2.4. Genetic analysis

DNA was extracted from blood lymphocytes by standard

techniques after obtaining informed consent. The following

microsatellite markers were studied to determine potential

linkage to known CMD loci: D6S407, D6S1705 and

D6S1620 for LAMA2; D9S306, D9S2105, D9S2171 and

D9S2107 for FCMD; D1S211, D1S2677, D1S197 and

D1S200 for MEB; D1S2871, D1S213, D1S2833 and

D1S459 for MDC1B [3,4,7,11,12].

In all cases the forward primer was labelled at its 5 0end by

a 6-FAM, NED (or TET), or HEX fluorochrome. PCRs were

performed under the following conditions: 40 ng genomic

DNA, 1 £ buffer supplied by the manufacturer (Perkin–

Elmer), 0.15 mM of each dNTP, 5 pmol of each primer,

and 0.5 U AmpliTaq Gold polymerase (Perkin–Elmer) in

a final volume of 15 ml. The amplification conditions were

S. Quijano-Roy et al. / Neuromuscular Disorders 12 (2002) 466–475 467

10 min denaturation at 948C; then 30 cycles with 30 s at

948C, 30 s at 558C, 1 min at 728C; and finally an extension

step at 728C for 3 min. Amplified PCR products were sepa-

rated by electrophoresis on a 4% acrylamide/bisacrylamide

19:1 and 6 M urea gel, using a 377 DNA sequencer (Applied

Biosystems). Results were analysed by GENSCAN (version

3.1) and GENOTYPER (version 2.1) software.

3. Results

3.1. Clinical features

The clinical features of the series are described below and

summarised in Table 1. Severity and precocity of the disease

was variable. The two first cases initially showed mild but

consistent congenital hypotonia. They were able to walk for

a few years and the disease progressed subsequently during

the first decade. Cases 3 and 4 were severe from early

infancy and never walked. Despite this apparent heteroge-

neity, all of them showed a strikingly similar phenotype at

advanced stages of the disease (second decade in patients 1

and 2, end of the first decade in patients 3 and 4). All the

patients were tetraplegic, mechanically ventilated, showed a

giant macroglossy and had extremely wasted hands and feet

(Figs. 1 and 2). While CK levels initially were very high in

all cases, they decreased subsequently to normal values in

early adulthood.

3.1.1. Patient 1

This patient was the first child born to French non-consan-

guineous parents and had a healthy sister. Her birth weight

was 3480 g. Mild neonatal hypotonia was reported since the

first year when interviewing the family retrospectively.

Motor milestones were slightly delayed. Sitting position

was unsteady at 7 months, and walking was possible at 17

months. At 2 years she was referred for evaluation because of

frequent falls and walking difficulties. On examination, the

patient had lumbar hyperlordosis, hypertrophic calves and

proximal weakness revealed by a waddling gait and a posi-

tive Gower’s sign. At 3 years marked lumbar hiperlordosis

and wasting of the proximal muscles of upper limbs were

noticed. In the following years, the patient showed a dramatic

progression of the amyotrophy and weakness, first affecting

the axial and proximal musculature and subsequently the face

and distal limbs. A rigid corset was required at the age of 6

years due to a rapidly progressive lumbar dorsal scoliosis.

She progressively lost all her motor abilities: she was not able

to walk after age 7, lost sitting position at 10, and needed head

support from age 13. At present she is 27 years old and is

S. Quijano-Roy et al. / Neuromuscular Disorders 12 (2002) 466–475468

Table 1

Clinical and paraclinical featuresa

Clinical features Patient 1 Patient 2 Patient 3 Patient 4

Maximal age follow up 27 years 19 years 15 years Dead at 10 years

Motor milestones

Congenital hypotonia 1 11 111 111

Maximal acquisition Walking (17 months) Walking (3 years) Sitting (12 months) Sitting (5 years)

Lost walking 7 years 6 years Never Never

Lost sitting 10 years 15 years 7 years 8 years

Lost head control 13 years 16 years 8 years 8 years

Contractures Second decade Ankles (9 years) Early Early

Macroglossia 111 111 111 111

Calf hypertrophy 11 11 111 11

Distal amyotrophy Second decade Second decade First decade First decade

Spinal deformities 111 11 11 11

Feeding difficulties Late Late Late Early

Gastrostomy 0 0 0 9 years

Respiratory insufficiency 111 111 111 111

Nasal noct. ventilation 12 years 13 years 10 years Refused

Tracheotomy 13 years 17 years 1 Refused

Diaphragmatic failure 111 11 1 1

Cardiac dysfunction 1 (25 years) N 1 (15 years) N

CK 111 (3 years) 111 (1 year) 111 (9 years) 111 (18 months)

Neurologic abnormalities

Mental retardation 0 0 ^ ^

Speech difficulties Late Late Early Early

Seizures 0 0 0 0

Brain MRI Mild atrophy Mild atrophy N Mild atrophy

Evoked potentials

Auditory N N N N

Visual Mild delay ^ N Nd

Somatosensory (wrist) Mild delay N N Nd

a Symbols: N, normal; 0, absent; ^, borderline or minimal; 1, mild; 11 , moderate; 111, severe; Nd, no data available.

capable only of antigravity movements in some fingers and of

some facial movements such as eye closure. Joint contrac-

tures were never significant until loss of ambulation. Respira-

tory function was also dramatically impaired around puberty.

Vital capacity (VC) dropped from 70% of the predicted

values at 9 years, to 40% at 11 years. At age 12 her VC

was 25% and a tracheostomy was required. At that time

there were signs of mild cardiac dysfunction with an ejection

fraction of 42% (normal of 60 ^ 10%). Repeated cardiac

studies in the following years did not reveal any significant

worsening of the left ventricle function (ejection fraction of

47% at 25 years). No right or left ventricle enlargement was

ever observed, and ECG and holter recordings were normal.

Macroglossia was initially noticed during puberty and

S. Quijano-Roy et al. / Neuromuscular Disorders 12 (2002) 466–475 469

Fig. 1. Clinical phenotype of patients 2 (a and b), 3 (c and d) and 4 (e and f). (a) Patient 2: note generalised increase in muscle bulk, with calf and quadriceps

hypertrophy at 3 years of age. (b). Patient 2: diffuse amyotrophy and macroglossia were evident at 17 years. (c) Patient 3: calf hypertrophy, ankle and hip

contractures were manifest at 7 years. (d) Patient 3: only minimal distal movements in the fingers remained possible at 12 years. Note the typical hand aspect

and macroglossia at that time. (e) Patient 4: maximal motor performance, sitting position, was maintained until the age of 8 years. (f) Patient 4: advanced severe

stage at 10 years: tetraplegy, giant macroglossia preventing mouth closure and deglutition.

progressed dramatically during the second decade. Despite a

partial glossectomy at age 24, the tongue continued to

enlarge, interfering severely with speaking, chewing and

swallowing. She was not mentally retarded. At 25 years audi-

tory evoked potentials were normal, visual and somatosen-

sory responses were slightly delayed, and brain MRI had only

mild frontal atrophy with no white matter or structural

abnormalities. Diagnosis of a muscle disorder was initially

suspected due to ten-fold increased CK levels (1800 UI/l)

and myopathic features on EMG studies at three years of

age. Muscle biopsies performed at 7 and 19 years showed

definite dystrophic changes with dramatic drop in the number

of fibres in the second biopsy. In a third sample taken from

paraspinal muscles at 25 years all muscle fibres had been

replaced by fatty tissue. Congo red staining excluded the

presence of amyloid deposits in this last sample. CK levels

at 25 years were normal (65 UI/l).

3.1.2. Patient 2

This 19 year-old girl was the only child from a non-consan-

guineous Caribbean family. Two prior gestations resulted in

spontaneous abortions. She was a 33-week-gestation prema-

ture infant and presented at birth symptoms of neonatal infec-

tion and mild transitory respiratory distress that did not

require resuscitation manoeuvres or mechanical ventilation.

At 14 months, in the context of a respiratory infection, she

was hospitalised and found to be hypotrophic, hypotonic and

weak. She was not able to walk without support until 3 years,

and never ran. Onset of calf hypertrophy was observed at 3

years, associated with generalised muscle pseudohypertro-

phy (Fig. 1a). Motor abilities deteriorated significantly in the

course of the following years. At 6 years she lost her ability to

walk and by 15 years she could not sit without support.

Macroglossia became evident around puberty, interfering

with speaking and eating (Fig. 1b). She required ankle

surgery at age 9 years due to important distal joint contrac-

tures. At 11 years, progressive scoliosis was diagnosed and

treated by a rigid corset until age 14 years when vertebral

arthrodesis was performed. With regard to the respiratory

function, VC began to deteriorate at 7 years and dropped to

24% of the expected values at age 13. Polysomnographic

studies revealed sleep hypoventilation which required night

positive-pressure nasal ventilation. VC continued decreas-

ing; at 17 years, tracheostomy and continuous mechanical

ventilation became necessary. Complete cardiac evaluation

was normal. Mental development was normal. Brain MRI

performed at 15 and 18 years showed some degree of non-

progressive cortical–subcortical atrophy. Auditory and

somatosensory evoked potentials were normal. Visual

responses at 19 years showed normal latencies but morphol-

ogy appeared slightly impaired. Last follow-up at 19 years

showed tetraplegy and extreme diffuse amyotrophy, includ-

ing the above-mentioned particular hand aspect. She was not

able to make any antigravity movements except for facial

muscles involved in eye closure, and only minimal proximal

and distal movements on the horizontal plane were possible.

While very increased CK levels were observed initially

(5560 UI/l at one year), normal results were obtained at 19

years of age (210 UI/l).

3.1.3. Patient 3

This 15 year-old boy is the third child of a Sudanese

consanguineous family with two non-affected children. He

was born at term after a normal pregnancy and delivery.

Axial hypotonia was first noticed at two months. He

acquired sitting position at 12 months of age; no further

motor acquisitions were achieved except for crawling on

his buttocks which remained possible until 5 years of age.

He was never able to stand or walk. Calf hypertrophy was

manifest at 3 years Muscle weakness, was initially predo-

minant in axial and proximal musculature and progressed

subsequently leading to loss of sitting position at 7 years.

Lumbar hyperlordosis and contractures in the lower extre-

mities were marked at 5 years. Progressive trunk weakness

required a rigid corset at 6 years. Subsequently, a head

support was necessary due to severe neck weakness. At 7

S. Quijano-Roy et al. / Neuromuscular Disorders 12 (2002) 466–475470

Fig. 2. Typical hand aspect; patients 2 (a) and 3 (b). Note the global atrophy and the hand rest attitude in supination and ulnar deviation; thumbs are in adduction

and partial opposition, while the fingers tend to permanent flexion.

years, macroglossia was firstly noticed. This seemed to

impair speech and feeding. At end-stages, around 10–11

years of age, amyotrophy was severe and only the distal

upper limbs remained minimally functional with partial

antigravity movements (Fig. 1c). Onset of restrictive

respiratory insufficiency was diagnosed at 5 years of age.

Clinical and radiological signs of diaphragmatic involve-

ment were subsequently observed, and a tracheotomy was

required at 11 years when VC was 25% of the theoretical

values. At this age, the patient received digoxin treatment

for one year because of transient cardiac insufficiency trig-

gered by a pulmonary infection. An echocardiography

performed at 13 years revealed mild cardiac abnormalities

consisting of a hypokinetic left ventricle with slightly

reduced shortening fraction (27% for a normal value over

30%) and systolic index (32% for a normal over 35%); the

ejection fraction was normal (61%). Treatment with an

angiotensin-converting enzyme inhibitor was then insti-

tuted; no clinical abnormalities or worsening of the echo-

cardiographic parameters were afterwards observed.

Cognitive function was interfered with by behavioural and

speech troubles. Although macroglossia impaired speaking

abilities, they existed prior to the tongue enlargement.

Evoked potentials showed no abnormalities at age 7. CK

levels were increased at 9 years of age (2270 UI/l). A

muscle CT scan performed at 15 years revealed severe

widespread replacement of muscle by fat-density tissue,

with only a minimal detection of muscle density at the ante-

rior compartment of the legs and extensors of the forearms.

3.1.4. Patient 4

This French girl was the third child of a non-consangui-

neous family with no other affected members. She was

slightly premature and presented significant hypotonia and

feeding difficulties since birth. CK was extremely high (40-

fold times the normal level) and a muscle biopsy at 6 months

showed dystrophic features. She was able to sit at 18

months, but sitting position remained unsteady until 5

years (Fig. 1e). She could never stand or walk. Her language

was also delayed and was never able to speak properly. She

developed progressive enlargement of the tongue by age 5–

6 years which interfered significantly with deglutition and

speech and eventually prevented the patient from mouth

closure. Since the language difficulties existed previously,

they seem not to be explained only by the tongue enlarge-

ment. She also presented delay in acquisition of sphincter

control. These findings suggested a certain degree of intel-

lectual dysfunction, but the existence of definite mental

retardation remained unclear. Around 3–4 years, calf hyper-

trophy was evidenced. In addition, moderate kyphosis and

diffuse joint contractures were observed. At 7–8 years,

progressive and diffuse muscle wasting was accompanied

by deterioration of her motor abilities and extreme weak-

ness. She lost sitting ability and required head support by 8

years. A lower limb CT scan revealed substitution of muscle

tissue by fat-density tissue, especially in the axial and prox-

imal musculature. Distally, gastrocnemius and soleus

muscles were also severely affected. Muscles of the anterior

aspect of the leg were comparatively well preserved. After a

few years, the patient lost almost all antigravity movements,

except for a minimal function in her fingers. End-stage

phenotype was characterised as in the prior cases by severe

generalised amyotrophy with extreme wasting of hands and

feet. A gastrostomy was required at 9 years due to feeding

difficulties increased by the severe tongue enlargement (Fig.

1f). She had difficulty in emptying her bladder but no

evidence of an obstructive cause was detected. Restrictive

respiratory insufficiency with clinical and radiological signs

of diaphragmatic involvement was developed and

progressed dramatically in a few years. VC dropped from

70% of its theoretic value at 4 years to 14% at 9 years. The

patient’s family refused tracheotomy and she died at 10

years due to respiratory complications. No symptoms or

signs of cardiac involvement were ever detected. A brain

MRI performed at 9 years had revealed only mild cortico-

subcortical atrophy, without significant white matter

changes. Auditory evoked potentials were unremarkable.

3.2. Morphological features

3.2.1. Light microscopy

All muscle biopsies showed an important variation in

fibre size, with mild increase in the frequency of internal

nuclei, a severe augmentation of interstitial connective

tissue and a marked predominance of type 1 fibres (Fig.

3). Some rare necrotic and regenerating fibres were present.

The muscle biopsies performed at 25 and 19 years from

Patients 1 and 2, respectively, showed extensive fibro-

adipous replacement with hardly any fibres left. These find-

ings are compatible with the progressive character of the

disease. In patient 1, whose last biopsy was analysed

using the Red Congo staining, no accumulation of amyloid

material was detected.

3.2.2. Inmunofluorescence

Immunocytochemical results are summarised in Table 2;

Fig. 4. All muscle biopsies showed overexpression of lami-

nin a5 and reduced expression of laminin a2 chain with an

antibody that recognises the 80 KDa fragment. Cases 2 and

3 showed reduced expression of laminin a2 with an anti-

body that recognises the 300 KDa fragment, as well as

virtual absence of a-dystroglycan expression. No muscle

was available in cases 1 and 4 to study the expression of

these proteins and of others that were normal in patients 2

and 3 (b-dystroglycan ,sarcoglycans b and g, and laminins

b1 and g1). Dystrophin and a-sarcoglycan were normally

expressed in all cases.

3.3. Genetic analysis

Several polymorphic markers spanning the LAMA2 (6q2),

FCMD (9q31-q33), MEB (1p32-p34) and MDC1B (1q42)

loci were studied for the four cases. The genetic analysis

S. Quijano-Roy et al. / Neuromuscular Disorders 12 (2002) 466–475 471

ruled out the four loci in the only consanguineous family

(case 3), based on the absence of homozygosity of the

haplotypes transmitted to the affected child [13]. Further-

more, this patient shared identical haplotypes with his unaf-

fected brother for FCMD and MEB loci.

A mutation in the FCMD gene, fukutin, was excluded for

case 4 by direct sequencing of the whole gene (Toda, perso-

nal communication).

4. Discussion

In this study we present four non-related children, three

females and a male, affected with a form of CMD with a

distinct phenotype. Since all parents were healthy and

consanguinity was present in one case, a recessive autoso-

mal inheritance is probable.

The clinical features shared by the four patients, espe-

cially at advanced stages of the disease, strongly suggest

that they constitute a single entity. They all had an

‘atrophic–hypertrophic’, progressive phenotype. Calf

hypertrophy and proximal weakness were observed in the

first years of life. Later features were progressive weakness,

severe restrictive respiratory insufficiency, macroglossia,

extreme diffuse weakness and amyotrophy with a peculiar

aspect in hands. All the patients presented facial weakness

and a round and ‘puffy’ aspect of the face, but it was always

a late finding, especially in the two milder cases. Although

all showed a similar course, some particularities were

noticed. Cases 1 and 2 presented initially a mild phenotype

and were able to walk without support for a few years. They

did not develop significant joint contractures or facial invol-

vement during the first decade. In contrast, cases 3 and 4

showed more severe early signs and never walked. On the

other hand, although there were no major central nervous

S. Quijano-Roy et al. / Neuromuscular Disorders 12 (2002) 466–475472

Table 2

Immunohistochemistry resultsb

Antibodies Patient 1 Patient 2 Patient 3 Patient 4

Dystrophin COOH terminal N N N N

a-sarcoglycan N N N N

a-dystroglycan Nd 0 0 Nd

Laminin a2 (80 KDa) d d d d d

Laminin a2 (300 KDa) Nd d d 0 Nd

Laminin a5 b b b b b b

Laminin g1 Nd N N Nd

Laminin b1 Nd N N Nd

a Symbols: N, normal expression; d , mild diminution; d d , important

diminution; 0, absent; b , overexpression; b b , important overexpres-

sion; Nd, non-determined.

Fig. 3. Transversal sections of muscle biopsies from patient 2 (a), patient 3 (b), patient 4 (c) and patient 1 (d) at the ages of 19, 15, 8 and 7 years respectively. All

show a similar dystrophic pattern consistent with CMD, with extensive fibroadipous replacement of muscle fibres. HE, 20 £ .

system symptoms in any of the patients, cases 3 and 4 had

both significant speech delay and certain reasoning difficul-

ties that could suggest a mild cognitive dysfunction. Since

macroglossia and facial weakness were late features they do

not easily explain these early speech difficulties. In contrast

to the different intellectual performances, no major differ-

ences were found on neuroimaging studies; indeed, no

major structural or white matter abnormalities were found

in any of the four cases. A mild cortical–subcortical atrophy

was detected in cases 1, 2 and 3, but it is a non-specific

S. Quijano-Roy et al. / Neuromuscular Disorders 12 (2002) 466–475 473

Fig. 4. Immunofluorescence labelling of two different muscle samples from patient 2, obtained at 7 years (a and b) and 19 years of age (c–f); note the relative

preservation of muscle structure in the first sample compared with the second one, in which only scarce fibres, surrounded by fibrous tissue, were found. (a)

Normal expression of a-sarcoglycan. (b) Laminin a5 was normally expressed in blood vessel walls and overexpressed in muscle fibres. A partial deficiency in

laminin a2 expression was observed both with MAB1922 (c) and with NCL-merosin (d) antibodies; note the normal merosin staining of a blood vessel in the

lower right part of (d) a-Dystroglycan was virtually absent from all fibres (e), while b-dystroglycan was normally expressed (f). Transversal sections; 40 £ in

(a and b), 20 £ in (c–f).

finding, also observed in chronic hypoxia. All cases

presented grossly elevated serum CK levels in the early

stages of the disease that decreased to normal in advanced

stages of the disease, probably due to the severe amyotro-

phy. The immunohistochemical pattern was also homoge-

neous, the most outstanding features being a reduced

expression of the laminin a2 chain coexisting with up-regu-

lation of laminin a5 in all cases, and a severe reduction of a-

dystroglycan expression in the 2 cases tested. This consis-

tent pattern of immunofluorescence supports the idea that

these patients may constitute a single entity.

Considering this series of patients a congenital muscle

disorder may be controversial. In fact, the four patients

share clinical features of congenital and non-congenital

muscular dystrophies. They presented early hypotonia

and/or weakness and delayed motor milestones, as seen in

CMDs. Congenital hypotonia in case 1 was mild and only

revealed retrospectively when interviewing the family. On

the other hand, progressive loss of prior motor acquisitions

in the course of a few years and calf pseudohypertrophy are

more typical of non-congenital, ‘progressive’ muscular

dystrophies such as dystrophinopathies and sarcoglycano-

pathies. Immunocytochemical studies and atypical clinical

features excluded this possibility. On the other hand, the

concurrence of macroglossia, muscle pseudohypertrophy

and diaphragmatic failure is a known typical feature of

amyloid myopathy [14,15] in which a preferential accumu-

lation of amyloid occurs in tongue, diaphragm and fatty

tissue. Although no tongue or diaphragm samples were

studied in our series, red Congo staining of a paraspinal

muscle specimen in patient 1 did not show any amyloid

deposits. In addition, no other clinical features suggesting

this disease were observed in any of our patients.

Finally, the patients described here did not show the

diffuse white matter changes or the rather static course

with early joint contractures observed in merosin-deficient

CMD patients with defects in the LAMA2 gene. Moreover,

linkage analysis ruled out the LAMA2 locus on chromosome

6q22 suggesting that merosin deficiency is a secondary

phenomenon.

Among the CMDs without central nervous system invol-

vement, three forms with secondary merosin deficiency and

a-dystroglycan reduction have been published recently

[8,9,16]. A severe form of CMD, MDC1B [7,8], associated

with calf hypertrophy and diaphragmatic failure, could be

suspected in our patients. MDC1B patients presented early

facial weakness and rigidity of the spine, and were found to

remain stable except for the respiratory symptoms.

However, their follow up was relatively short, the oldest

case being only 11 years old at the time of the report.

Linkage to the putative MDC1B locus in chromosome

1q42 was ruled out in our only consanguineous patient

(case 3). Secondly, Mercuri et al. [16] have reported two

Scottish siblings with a homogeneous phenotype charac-

terised by early feeding difficulties due to bulbar and facial

weakness, progressive course and joint contractures. The

older patient died suddenly at 7 years and no calf hyper-

trophy or significant respiratory insufficiency had been

detected up to that moment; however, her 14-month-old

sister had mild calf and quadriceps hypertrophy. Although

some of the late clinical features typical of our patients

were not present in these two forms, their up-to-now

short evolution does not allow us to exclude further simi-

larities. Another family with generalised muscle hypertro-

phy, very high CK levels and partial merosin deficiency

showing almost complete depletion of a-dystroglycan has

been briefly and recently reported by T. Voit [9]. In

patients 3 and 4, a form of CMD with associated brain

involvement could be suggested by some clinical findings.

FCMD or MEB-disease was initially suspected in these

patients. However, brain MRI does not show structural

changes of the posterior fossa or cortical dysplasia, and

neither structural ocular abnormalities nor severe myopia

were observed. Moreover, genetic studies excluded invol-

vement of the FCMD gene, fukutin, in these two patients,

and linkage to MEB locus in patient 3. The combination of

calf hypertrophy, severe amyotrophy and mental retarda-

tion in the absence of CNS structural abnormalities has

been reported by Topaloglu et al. in two Turkish siblings

[17]. The genetic basis of this form is yet unknown, but

lack of progression at 6 and 10 years, early facial involve-

ment and especially severe mental retardation distinguish

this phenotype from our patients. Two Italian patients have

been presented by Villanova et al. [18] suffering from a

secondary merosin-deficient form with calf hypertrophy,

macroglossy and patchy periventricular white matter

lesions; a-dystroglycan was severely depleted in muscle.

However, the patients presented profound mental retarda-

tion, cerebellar hypoplasia, and severe myopia. They had a

quite static course and did not develop respiratory symp-

toms.

Future molecular characterisation of the different pheno-

types described above will be critical in the identification of

distinct CMD forms and better precision of their clinical

spectrum.

Reduction of a-dystroglycan in muscle, recently

described in some of the secondary merosin-deficient

forms of CMD mentioned above [9], is a finding of uncer-

tain significance. We found a strikingly severe depletion of

this protein in the two patients in whom it was studied. This

could be due to a primary abnormality of this protein, or to

secondary defects of the proteins involved in the stability of

the extracellular matrix, or to its binding to the DAG

complex in the muscular fibre.

To conclude, we propose that the four cases presented

here suffer from a particular and severe form of CMD char-

acterised by progressive weakness, calf pseudohypertrophy,

macroglossia, respiratory failure with diaphragmatic weak-

ness and extreme distal amyotrophy, but without significant

abnormalities in brain MRI and coexisting with severe

depletion of a-dystroglycan and secondary partial defi-

ciency of the laminin a2 chain in muscle. Further investiga-

S. Quijano-Roy et al. / Neuromuscular Disorders 12 (2002) 466–475474

tions are necessary to determine the genetic basis of this

novel entity and to establish its possible relationships with

other forms of CMD.

5. Addendum

Investigation of a new candidate gene, the fukutin related

protein (FKRP) has led to the identification of mutations in

the four patients presented in this publication (Brockington

et al., Am J Hum Genet 2001;69:1168–1209).

Acknowledgements

We wish to thank the patients and their families for their

co-operation. We also acknowledge the help of Dr Louis

Viollet (Garches) for the muscle biopsies, Huguette Collin,

Martine Chevallay (INSERM U523), Veronique Levy and

Isabelle Le Maner (Faculte de Medecine Paris-Ouest) for

the immunofluorescence studies and Dr Fernando Tome

(INSERM U523) for critical reading of the manuscript.

We gratefully acknowledge the financial support of the

Association Francaise contre les Myopathies (AFM,

France), INSERM (French INSERM/AFM Research

network on rare disorders) and the European Commission

(Myocluster No. QLG1-1999-00870).

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